Valved, microwell cell-culture device and method

ABSTRACT

A valved microfluidics device, microfluidics cell-culture device and system incorporating the devices are disclosed. The valved microfluidics device includes a substrate, a microchannel through which liquid can be moved from one station to another within the device, and a pneumatic microvalve adapted to be switched between open and closed states to control the flow of fluid through a microchannel. The microvalve is formed of three flexible membranes, one of which is responsive to pneumatic pressure applied to the valve and the other two of which deform to produce a more sealable channel cross-section. The cell culture device provides valving to allow controlled loading of cells into the individual well of the device, and exchange of cell-culture components in the wells.

This patent application is a continuation of U.S. patent applicationSer. No. 13/602,328 filed Sep. 4, 2012, (now U.S. Pat. No. 8,673,625,issued Apr. 11, 2013) which is a divisional of U.S. patent applicationSer. No. 11/648,207 filed Dec. 29, 2006 (now U.S. Pat. No. 8,257,964,issued Sep. 4, 2012), which claims priority to U.S. provisional patentapplication No. 60/756,399 filed on Jan. 4, 2006, which is incorporatedin its entirety herein by reference.

FIELD OF THE INVENTION

The present invention relates to a microwell device and method, and inparticular, to a valved microwell array device designed for highthroughput cell culture assays, to microvalve for use in the device, andto methods for making the valve and device.

BACKGROUND OF THE INVENTION

There is a growing demand in the drug discovery and related fields forhigh throughput cell culture systems, that is, systems capable ofsupporting large numbers of cell-culture assays in parallel. For avariety of reasons, it would be desirable to conduct large-scalecell-culture assays in a microfluidics device having an array ormicrowells and microfluidics structure for populating and feeding thewells. One major advantage of microfluidic cell culture is thepossibility to mimic in vivo conditions. Culture parameters such asmedium flow rate, shear stress, Peclet number, Reynolds number,liquid/cell volume ratio, length scale, and cell density can becontrolled to more closely match physiologic conditions. Continuousmedium perfusion and “on-chip” monitoring ensure a stable environmentfor cells during observation. These factors should limit variations incell behavior and improve the statistical power of experiments. It isalso likely that by providing more in vivo-like culture conditions, cellbehavior will be closer to physiologic conditions, making assay resultsmore relevant for medical applications.

The potential advantages of a microwell array device have been realizedto a rather limited extent only in the prior art. Various limitationsassociated with prior art devices include (i) the requirement for bulkyrobotics to populate the wells in the device, (ii) difficulty inpreventing microfluidics structures from being blocked by cell growthwithin the structures, (iii) inability to sustain uniform cultureconditions over an extended assay period, (iv) inability to achieve andalter cell-culture conditions at the level of individual wells, and (v)difficulty in creating the necessary microfluidics structuresefficiently by microfabrication.

It would therefore be desirable to provide a microwell array devicecapable of more fully realizing the advantages noted above in a highthroughput cell culture system. There is also a need to achieve theseadvantages in a microfluidics device that can be constructed efficientlyby microfabrication.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a valved microwell device composedof a substrate having formed therein, a microfluidics passageway having(i) a microwell for receiving particles, such as cells therein, (ii) aninlet channel segment, (iii) an outlet channel segment, (iv) a channelintersection segment having a first channel arm that communicates theinlet and outlet channel segments, and a second channel armcommunicating the inlet channel segment with the interior of the well,where the first channel arm flows around a portion of the well, and (v)a porous barrier through which fluid, but not cells, in the well canperfuse from the well into the first arm.

A microvalve disposed in the second channel arm is operable to controlthe flow of fluid from the inlet channel segment into the well, suchthat a cell carried in a fluid moving through the inlet segment can bediverted into the well by opening the valve, and once diverted into thewell, can be captured therein, with fluid flowing through thepassageway, by closing the valve, and culture medium flowing through thefirst arm can exchange solute components with culture medium within thewell by diffusion of such components across the porous barrier. Themicrovalve may have the construction described below.

The device may further include another microvalve disposed in the firstchannel arm, operable to control the flow of fluid from the inletchannel to the outlet channel. The first channel arm may have a pair ofbranches that flow around opposite sides of the well, and the othervalve may include a pair of valves controlling fluid flow through eachbranch.

The device may include a plurality of such passageways, a microchannelwell-distribution network for supplying input fluid to each of aselected one or more of wells in the passageways, under the control of aplurality of valves associated with the network, and first and secondvalve-supply networks for supplying fluid pressure to the first andsecond microvalves, respectively.

In still another aspect, the invention includes a microarray culturesystem, including a microarray device having a substrate, and formed inthe substrate, a plurality of microfluidics passageways, each having (i)a well for receiving particles, such as cells therein, (ii) an inletchannel segment, (iii) an outlet channel segment, (iv) a channelintersection segment having a first channel arm that communicates theinlet and outlet channel segments, and a second channel armcommunicating the inlet channel segment with the interior of the well,where the first channel arm flows around a portion of the well, and (v)a porous barrier through which fluid, but not cells, in the well canperfuse from the well into the first arm. Associated with eachpassageway is a first microvalve disposed in the first channel arm forcontrolling the flow of fluid from the inlet channel segment into thefirst channel arm, and a second microvalve disposed in the secondchannel arm for controlling the flow of fluid from the inlet channelsegment into the passageway well.

A microchannel well-distribution network in the device is operable tosupply input fluid to each of a selected one or more of wells in thepassageways, under the control of a plurality of valves associated withthe network, and first and second microchannel valve-supply networks areoperable to supply fluid pressure to first and second valves in thepassageways, respectively. A plurality of reservoirs is each in fluidcommunication with a channel in the well-distribution network or in thevalve-supply network. A controller in the system operates for supplyingpressurized fluid to selected ones of the reservoirs, thereby to supplyfluid to a selected one or more of the microfluidic passageways and toselected valves.

In a system containing N passageways, the microchannel distributionnetwork may have X separate valved channels, where X=2 log₂N. The firstand second valve-supply networks may operate to supply fluid pressuresimultaneously to all of the first valves, and to all of the secondvalves, respectively.

The system may further include a detector by which the presence orabsence of cells in a passageway intersection can be determined, and acontroller for sequentially activating the second and first valves forcapturing a cell in such intersection in the associated well.

The reservoirs in the system may have at least one cell reservoir forholding cells to be introduced into the passageways, and at least onereagent reservoir for holding a solution to be perfused through thepassageways. The system may further include a sample-control network forcontrolling the flow of fluid in the cell and reagent reservoirs to thewell-distribution network.

The controller may operate in one mode to open the first valve, andclose the second valve in each passageway, so that a cell-culture mediumcontained in a reagent reservoir flowing from a device inlet to a deviceoutlet can exchange solute components with cell culture medium in eachwell across the porous barrier.

The controller may operate in another mode to close the first valve, andopen the second valve of in each passageway, so that a medium flowingfrom a device outlet to a device inlet will carry the cells in thedevice wells out of the device.

In another aspect, the invention includes a valved microfluidics devicehaving a substrate, a microchannel through which liquid can be movedfrom one station to another within the device, and a pneumaticmicrovalve adapted to be switched between open and closed states tocontrol the flow of fluid through a microchannel. The microchannel isformed of (i) two or more flexible membranes forming wall portions in avalved region of the microchannel, including a primary membrane and oneor more secondary membranes that are each joined to the primary membraneat a common edge, and (ii) a chamber formed in the substrate, separatedfrom the microchannel by the primary membrane and adapted to receive apositive or negative fluid pressure, thus to deform the primarymembrane. Deformation of the primary membrane causes the secondarymembrane(s) to deform, and the combined deformation of the primary andsecondary membranes is effective to switch the condition of the valvebetween its open and closed states.

The device may include, for each secondary membrane, a recess formed inthe substrate into which the secondary membrane is deflected whendeformed. The flexible membranes may include a top-wall primary membraneand a pair of opposite side-wall secondary membranes, with the valvedregion of the microchannel, with the valve in its open state, beingsubstantially rectangular. The height to width ratio of the rectangularmicrochannel may be at least about 0.5 to 1.0. The application ofpositive pressure to the chamber, to place the valve in its closedstate, may cause the primary membrane to bow outwardly into the channel,and the secondary membranes to bend outwardly at their common edges withthe primary membrane, into the associated recesses, thus to enhance theextent of sealing between the primary membrane and the two secondarymembranes as the primary membrane is deformed.

The flexible membranes in the device may be formed of any elastomer thatis compatible with microfabrication techniques, e.g., PDMS elastomer.

The microchannel in the device may intersect a channel segment, and thevalve may be positioned in the channel segment to control the amount offluid flow from the microchannel into the segment. The channel segmentmay connect the microchannel with a well formed in the substrate, wherethe valve is used to control the flow of fluid from the micro channelinto the well.

In a related aspect, the invention includes a microfluidic devicecomprising an elastomeric monolith situated between a rigid substrateand a semi-rigid substrate. In one embodiment the two substrates areplanar. In another embodiment the elastomeric body comprises multipleelastomeric layers separately prepared and having defined in a firstsurface of each layer a pattern of channels and/or chambers. Theseparate layers are bonded together to form the monolith such that themicrofluidic features (e.g. channels, chambers, etc.) defined in thesurface of one layer are sealed off against the surface that lacksmicrofluidic features of a different layer. Preferably, the differentlayers contain features that operate in conjunction with those ofanother layer. When bonding the layers, the features of each layer arealigned such that they operate in conjunction with one another.Preferably, an adhesion promoter is used to bond the semi-rigidsubstrate to the elastomeric monolith.

In one embodiment the microfluidic device is prepared by combining afirst elastomeric layer having microfluidic channels in a first surfaceand a second elastomeric layer having pneumatic control chambers in afirst surface to form an elastomeric monolith. The monolith ischaracterized by having microfluidic channels disposed in a firstsurface and pneumatic control chambers disposed within the body of themonolith. Access holes providing communication from the external worldto the channels and chambers are also provided through both of thesubstrates and the monolith.

In yet another aspect, the invention includes a method for fabricating avalved microfluidics device of the type described above. The methodincludes, in part, the steps of preparing a mold having a fluorocarbonsurface coating, dispensing an elastomeric precursor over the coatedmold; at least partially curing the elastomeric precursor and removingthe at least partially cured elastomer from the mold. The method alsofurther includes, in part, placing the side of a semi-rigid sheet coatedwith an adhesion promoter on top of the elastomeric precursor prior tothe step of at least partially curing the elastomer, and after thecuring step, removing the joined semi-rigid sheet and elastomer from themold.

In one embodiment of a method for fabrication of a multilayermicrofluidic device, two molded elastomeric layers are formed, joinedtogether and bonded to a substrate. A first mold is prepared having anupper layer fluidic design, and a second mold is prepared having a lowerlayer fluidic design. Preferably, the molds have a fluorocarbon surfacecoating. After an elastomer precursor is dispensed over the first andthe second molds, the side of a semi-rigid sheet coated with an adhesionpromoter is placed on top of the elastomer precursor spread on the firstmold. The elastomer precursors are at least partially cured. Thereafterthe joined semi-rigid sheet/elastomer unit is removed from the firstmold, and the molded surface of the unit is placed over and aligned withthe elastomer residing on the second mold. The two elastomer surfacesare bonded together, and the elastomers are fully cured during orfollowing bonding. Thereafter the bonded elastomer assembly is removedfrom the second mold. The molded surface of the lower layer is bonded toa rigid substrate, thereby enclosing the features of the molded surfaceof the lower layer.

In yet another aspect, the invention includes a method of fabricating amicrofluidic device comprising (a) an elastomer monolith and (b) aholder for both securing the monolith and providing ports for addressingthe fluidic features of the monolith with external fluids and/orremoving fluids from the monolith. The fabrication method comprises (a)preparing a module comprising an elastomeric monolith having on one facea semi-rigid thermoplastic substrate adhered thereto, with ports throughthe semi-rigid substrate accessing a microfluidic network formed in theelastomeric monolith, (b) preparing a holder having on a first surfaceat least two recessed openings and having on the opposite surface acavity, and openings within the cavity communicating with each of therecessed openings, and (c) bonding the semi-rigid substrate of themodule within the cavity such that the ports of the module align withthe openings in the cavity.

These and other objects and features of the invention will become morefully understood when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a microarray cell culture system employing multiplemicrowell array devices constructed in accordance with the invention;

FIG. 2 shows one of the cell-culture array plates in the system of FIG.1 and the reservoir connections to the microarray device on the plate;

FIG. 3 shows a plan view of a microarray cell culture device having an8×2 well array, and channel connections to 18 reservoirs;

FIG. 4 shows the truth table for the microchannel well-distributionnetwork in the device of FIG. 3;

FIGS. 5A and 5B show microvalve channel connections to the well deviceof FIG. 3 (5A) and an enlarged view of a single well and associatedmicrovalves in the device;

FIGS. 6A and 6B illustrate of valve configuration of the FIG. 3 devicewhen cells are to be supplied to the wells in the device (6A), and whenthe cells are being directed to a specific well (6B):

FIGS. 7A-7E illustrate valve-switching steps employed in capturing acell in a well in the device, during cell loading to achieve selectednumbers of cells in each well (7A-7D) and components of an automatedcell loading assembly in the system;

FIG. 8 shows the condition of certain valves in the device when twodifferent drug solutions are perfused through wells 1-8 and 9-16,respectively, during a cell assay;

FIGS. 9A-9E are cross-sectional views of a microvalve device constructedin accordance with an embodiment of the invention, where 9D and 9E showthe valve membranes in open and closed conditions, respectively;

FIG. 10A-10H illustrate the condition of a microvalve such as shown inFIG. 9 with increasing fluid pressure applied to the valve;

FIG. 11 is a three-dimensional perspective view of a single well andassociated microvalves constructed in accordance with the invention, andhaving the layout of the passageway seen in plan view in FIG. 5B;

FIG. 12 is a three-dimensional perspective view of a three well andassociated microvalves and channel connections in the device of theinvention;

FIGS. 13A-13H illustrate successive steps for fabricating a mold usefulfor making a well, channels and valve region in a microfluidic layer ofthe microfluidic device of the invention;

FIGS. 14A-14E illustrate successive steps for fabricating a mold usefulfor making pneumatic chambers in a control layer of the microfluidicdevice of the invention;

FIG. 15 illustrates an acrylic sheet used in the preparation of themicrofluidic device of the invention;

FIGS. 16A-16K illustrate successive steps used to fabricate themicrofluidic device of the invention; and

FIGS. 17A-17D illustrate successive steps for preparing a device holderand an integrated microfluidic device/holder.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

A “particle” refers to biological cells, such as mammalian or bacterialcells, viral particles, or liposomal or other particles that may besubject to assay in accordance with the invention. Such particles haveminimum dimensions between about 50-100 nm, and may be as large as 20microns or more. When used to describe a cell assay in accordance withthe invention, the terms “particles” and “cells” may be usedinterchangeably.

A “microwell” refers to a micro-scale chamber able to accommodate aplurality of particles. A microwell is typically cylindrical in shapeand has diameter and depth dimensions in a preferred embodiment ofbetween 100 and 1500 microns, and 10 and 500 microns, respectively. Whenused to refer to a microwell within the microwell array device of theinvention, the term “well” and “microwell” are used interchangeably.

A “microchannel” refers to a micron-scale channel used for connecting astation in the device of the invention with a microwell, or a stationand a valve associated with the microwell. A microchannel typically hasa rectangular, e.g., square cross-section, with side and depthdimensions in a preferred embodiment of between 10 and 500 microns, and10 and 500 microns, respectively. Fluids flowing in the microchannelsmay exhibit microfluidic behavior. When used to refer to a microchannelwithin the microwell array device of the invention, the term“microchannel” and “channel” are used interchangeably.

A “microfluidics device” refers to a device having various station orwells connected by micron-scale microchannels in which fluids willexhibit microfluidic behavior in their flow through the channels.

A “microvalve” refers to a valve operable to open and close amicrochannel to fluid flow therethrough. When used to refer to amicrovalve within the microwell array device of the invention, the term“microvalve” and “valve” are used interchangeably.

A “microwell array” refers to an array of two or more microwells formedon a substrate.

A “device” is a term widely used in the art and encompasses a broadrange of meaning. For example, at its most basic and least elaboratedlevel, “device” may signify simply a substrate with features such aschannels, chambers and ports. At increasing levels of elaboration, the“device” may further comprise a substrate enclosing said features, orother layers having microfluidic features that operate in concert orindependently. At its most elaborated level, the “device” may comprise afully functional substrate mated with an object that facilitatesinteraction between the external world and the microfluidic features ofthe substrate. Such an object may variously be termed a holder,enclosure, housing, or similar term, as discussed below. As used herein,the term “device” refers to any of these embodiments or levels ofelaboration that the context may indicate.

B. Microarray Culture System and Device

FIG. 1 shows a microwell array system 20 in accordance with one aspectof the invention, for conducting cell-culture assays, and moregenerally, for conducting assays involving particles such as cells,viruses, liposomes, etc. in a culture environment. System 20 includes achamber 22 for holding a plurality of plates, such as plates 24,26,which will be described below with respect to FIG. 2. These plates arecarried on a cell-culture tower 28 having a plurality of plate slots,such as slot 30, for releasably holding individual plates. Each slotprovides a plurality of fluid pressure connections (not shown) betweenreservoirs in each plate and system solenoids, such as solenoids 31,33,which can be activated to supply pressurized fluid, such as air at 500kPa pressure, to the respective reservoirs in the plates supported ineach slot. In the embodiment that will be illustrated herein, themicroarray device in each plate contains an 8×2 array of microwells, andis serviced by a total of 18 reservoirs per plate. The system thuscontains 18 individually controllable solenoids, where each solenoidwill supply pressure to a 10-outlet manifold that connects that solenoidto a designated reservoir in each of the 10 plates.

A robotic arm 32 in the system is vertically shiftable on the tower topositions at which the arm can engage a selected plate, such as plate27, remove the plate from its slot, rotate the engaged plate 180°, andvertically move the plate for placement on a horizontally movable x-ystage 35 of a loading and observation structure 34 in the chamber. Whena plate is removed from a slot, and thus disconnected from the pressuresupply lines from the solenoids, it may be connected to a manifoldcoupler 37 which couples the plate reservoirs to the respectivesolenoids, allowing activation of various valving functions used forloading cells into the microwell array device carried on the plate, whenthe plate is positioned on structure 34, as will be described below.

Structure 34 includes a microscope 36, camera 38, and an opticaldetector 40 for sensing the position of cells at selected locations on amicrowell array chip supported on the plate, as will be described. Asnoted above, stage 35 is movable, in small x-y increments within thefiled of the microscope, to position the chip carried on the plate atselected located within the field of view of the microscope.

Culture conditions within the chamber are maintained by air- andCO₂-supply to the chamber and by heaters (not shown) within the chamber.

Also included in the system is a computer or processor 42, and keyboard46 and monitor 44 for user input and program display. The computer isoperatively connected to the detector and to the solenoids, such assolenoids 31, 33 for controlling gas pressure to the plate manifolds inaccordance with the cell-loading and cell-culturing operations performedby the system, to be described below.

FIG. 2 shows a plate, such as plate 24, for carrying out multiplecell-culture assays in parallel, in accordance with one aspect of theinvention. The plate illustrated supports a microarray device or chip 52having an array of microwells, microchannels, microvalves, and portstations, as will be described. Control of various media supplied to themicrowells, and microvalves, during a cell loading and culture assay,requires a plurality of reservoirs, such as reservoirs 48, 50,containing fluid which is delivered to a corresponding number of inputstations on the chip. The chip illustrated in FIGS. 2-6 has an 8×2 arrayof microwells, and is supplied by 18 reservoirs on plate 24. Eachreservoir is connected to a corresponding port station in the chip by achannel, such as channels 54, 56 formed on the plate.

In operation, the reservoirs in a plate are covered by a leak-tightgasket (not shown) that serves as a manifold between the systemsolenoids and each plate. That is, the gasket contains a line forpressurized gas between each solenoid manifold and one of the reservoirson the pressurized-gas line.

FIG. 3 shows the arrangement of microfluidic channels, well, and valvesin an 8×2 microwell array device 52 constructed according of anembodiment of the invention. The device shown contains an 8×2 array ofmicrowells, such as microwells 58, 60, where each microwell is part of apassageway, such as passageway 62 containing microwell 58. The layoutand operation of passageway 62 is described below with respect to FIG.5B. The chip is formed, in accordance with one aspect of the invention,by microfabricating the well, channel, and valve components on asubstrate 64 as will be detailed in Section D below.

Passageway 62 in the device, which is representative, is illustrated inenlarged layout view in FIG. 5B. The microwell 58 in the passagewayserves as one of the array of cell-culture microchambers in the device,and is supplied by an inlet segment 66 and drained by an outlet segment68 in the passageway. Specifically, inlet segment 66 terminates at anintersection 70, at which media can be directed either around the wellto segment 68 by a first 72 or second arm portion into the well, througha second arm 74. In the embodiment shown, the first arm includes a pairof first-arm portions 72 a, 72 b which bifurcate at intersection 70 andconverge at outlet 68. The circumferential portion of the microwell thatis bounded by arm portions 72 a, 72 b, is porous, or containsupper-ledge grooves, forming a barrier 76 effective to allow liquid inthe well, but not cells, to flow into or out of the well from the twoarm portions.

Also shown in FIG. 5B is a pair of first microvalves 78, 80 whichcontrol the flow of medium though arm portions 72 a, 72 b, respectively,and a second microvalve 82 which controls the flow of medium throughsecond arm 74. The construction and operation of the microvalves will bedetailed below in Section C. For purposes of the present discussion, itis only worth noting that the pair of second valves are activated bypressurized fluid supplied to the valves through a first microchannelvalve-supply network, seen locally at 84 in FIG. 5B, and that the secondvalve is activated by pressurized fluid supplied to the valve through asecond microchannel valve-supply network, seen locally at 86 in FIG. 5B.In operation, medium entering the passageway through inlet segment 66may be directed into microwell 58 by closing first valves 78, 80, andopening first valve 82. This mode is used in directing and capturingcells in the microwell. Once a desired number of cells are contained inthe well, the second valve is closed to capture the cells in the well,and the pair of first valves are opened to allow medium to flow througharm portion 72 a, 72 b, allowing exchange of medium components betweenthe arm portions and the well. Finally, after an assay is completed,cells may be removed from the wells, by opening the second valve,closing the two first valves, and forcing medium through each passagewayin an outlet-to-inlet direction.

As seen in FIG. 3 and FIG. 5A, the inlet segment in each passageway,such as inlet segment 66, is supplied by a microchannel, such asmicrochannel 87 that connects that passageway to a well-distributionnetwork indicated generally at 90 in FIG. 3. The outlet segment of eachpassageway, such as segment 68 in passageway 62, is drained by amicrochannel, such as microchannel 89, which feeds the passageway intoan outlet channel 118. Thus, each group of eight passageways in thedevice is supplied by eight microchannels arrayed directly above thepassageways, and each group is drained by eight microchannels, includingmicrochannel 84 arrayed directly below each group.

With particular reference to FIG. 3, device 52 has 18 port stations,such as station 88, at which fluid from one of the eighteen reservoirssupplying the device is delivered to the individual passageways in thedevice or to the valves controlling the movement of medium through thepassageways. These stations, which are numbered 1-18 in the figure,generally have the following supply or drain functions during chipoperations. Stations 1-3 supply cells (station 1) or one of twodifferent cell-culture media (stations 2-3) to each of the sixteenpassageways. This supply is effected by a three-inletsupply-distribution channel 92, also referred to herein as asample-supply network. From this channel, medium is directed to aselected passageway through the well-distribution network 90. As seen,the network has two sections 94, 96, each with eight microchannels, suchas microchannel 98 in section 94, that are connected to one of eightpassageways through a connecting microchannel, such as microchannel 87in section 96 supplying passageway 62.

Flow of medium from one of the three supply-reservoir stations to thewell-distribution network is controlled by a pair of microvalves 106,108 activated by fluid supply from station 18, and a pair of valves 104,105 activated by fluid supply from station 17.

Flow of medium through the well-distribution network is controlled bycoordinated activation of each of eight valve sets controlled by fluidsupply from stations 4-11. Each valve set, such as valve set 110,includes eight individual microvalves, such as microvalves 112, that arearranged on the sixteen channels of the network in a binary pattern seenin the truth table in Table 4. Columns 4-11 in this figure represent theeight valve-control stations, rows 1-16 represent the 16 passageways,indicated A1-H1 and A2-H2, and the unfilled blocks indicates amicrovalve at that position row and column in the network. The patternof filled blocks in the table indicates the pattern of closed valvesthat will direct medium to a selected one of the 16 passageways. Asseen, each passageway can be uniquely accessed by closing somecombination of four valves. For example, the microchannel supplyingfluid to passageway H1 has four microvalves at positions correspondingto stations 5, 7, 9, and 11. Thus, closing valves 4, 6, 8, and 10 willleave this channel free for fluid flow, while blocking all others.Similarly, the microchannel supplying fluid to passageway G1 has fourmicrovalves at positions corresponding to stations 4, 7, 9, and 11.Thus, closing valves 5, 6, 8, and 10 will leave this channel free forfluid flow, while blocking all others. More generally, employing thisbinary-control scheme, an array of N microchannels can be individuallyaccessed by X valve stations, each controlling N individual valves,where X=2 log₂N.

With continued reference to FIGS. 3 and 5A, reservoir stations 12-14 arestations through which media from one or more of the passageways aredrained from the chip into a corresponding plate reservoir. The threestations are connected to a three-outlet channel 118 having twomicrovalves 114, 116 along its length as shown. These microvalves arecontrolled by fluid supply from station 17, through a microchannel 120.

Completing the description of the device layout, and with reference toFIG. 5A, the pair of first microvalves in each passageway, such asmicrovalves 78, 80 in passageway 62, is activated by fluid supply fromstation 15, which supplies pressurized fluid to each valve pair througha microchannel 122 that branches into two supply microchannels 122 a,122 b. Thus supply of fluid pressure from station 15 is effective toclose each of the 16 pairs of first microvalves in the device.Similarly, each of the 16 second microvalves in the device is activatedsimultaneously by pressurized fluid from station 16, which suppliesfluid to each of the second valves through channels 124 and 126.

In a preferred embodiment, and as will be described more fully inSection C below, the device is preferably formed as a microfabricatedsilicon wafer, and has side dimensions of between about 50 to 150 cm.Each reservoir in plate 26 is designed to hold between about 0.001 and0.5 cc of fluid, e.g., liquid, and each microwell typically holds 1 to100 nl. The microwells and microchannels in the device have dimensionsas indicated above.

The following setup will illustrate plate preparation, cell loading, andincubation operations carried out in the system. For this illustration,it is assumed that three different media will be supplied to themicrowells: a suspension of cells used in loading cells into each of the16 microwells through station 1, and cell-culture media solutionscontaining two different drugs or different concentration of the samedrug, each of which will be supplied to one of the two groups of eightmicrowells (A1-H1 and A2-H2 in FIG. 4) through stations 2 and 3,respectively. These media are thus placed in reservoirs 1, 2, and 3 inplate 26 for delivery to the corresponding stations in the device. Allof the other reservoirs, which will supply fluid to stations 4-18 arefilled with a buffer solution. The device itself is placed on the plateso that its port stations are aligned to receive fluid from associatedreservoirs on the plate, as shown in FIG. 2.

After filling the plate reservoirs with the above fluids, the reservoirsare covered and sealed with a gasket manifold that serves to connecteach of the reservoirs to the associated solenoid valves. The plate isthen moved to stage 35 in the system for loading each microwell in thedevice with cells. This loading procedure is carried out successivelyfor each of the 16 microwells in the device. FIG. 6A illustrates thefirst valve setting required for cell loading. Here valves 104, 105 arein an open condition, and valves 106, 108 under the control of station18 are placed in a closed condition. When the cell-suspension medium isapplied under pressure to station 1, the cell suspension is forcedtoward each of the microchannel sections 94, 96, as indicated by theheavy fluid-path arrow in the figure. At the same time, valve 114, 116,are kept open to allow fluid in the passageways to be expelled throughstation 12 into the corresponding plate reservoir.

The device is now in a condition for introducing cells into each ofmicrowells. This is done, as indicated above, by selectively closingfour of the eight sets of valves under the control of stations 4-11, toallow passage of fluid through the network to one passageway only. FIG.6B illustrates the valve condition for supplying fluid through network90 to the passageway designed H1. Here, the valve sets controlled bystations 4, 6, 8, and 10 are closed, blocking flow of fluid through allof the microchannels in the network except the top one in the figureconnected the passageway H1. This valve configuration is maintained fora selected time and/or until a desired number of cells have entered andbeen captured in the selected microwell, as will now be described.

With continued reference to FIG. 6A, during the period that cells arebeing supplied to a selected microwell, the first and second microvalvescontrolling the movement of fluid in or around that well are controlledso as to achieve a desired number of cells in each well. In oneembodiment, for introducing a selected number of cells in each well,cell loading is carried out on a cell-by-cell basis, as illustrated inthe sequence of valve-control operation illustrated FIGS. 7A-7D. Inthese figures, a plate has been positioned on stage 35 in the system sothat a selected microwell, e.g., microwell 58 in passageway 62 is withinthe optical field of microscope 36. At the initial stage of thesequence, seen in FIG. 7A, the first valves are open and the secondvalve is closed, forcing cell in the passageway to travel around ratherthan into the microwell. As a cell moves toward the intersection in thepassageway, the first valves are closed and the second opened, as inFIG. 7B, allowing the single cell at the intersection to be carried intothe microwell. Once the cell is within the well, the second valve isclosed and the first valves opened, capturing the cell within the wellwhile a second cell moves into a capture position at the intersection(FIG. 7C). This sequence is repeated until a desired number of cellshave been captured in the selected microwell.

FIG. 6E shows components of the system control assembly for carrying outthe above cell-loading operations in an automated fashion. The positionof a cell in a microwell that is within the field-of-view of camera 38in FIG. 1 is being viewed. Where it is desired to fill each well with anapproximate, rather than precise number of cells, the above procedurecan be simplified simply by opening the second valve and closing thefirst valves in a selected passage for a given period of time, duringwhich all of the cells flowing through the passageway will be directedinto and captured in associated microwell.

In the above-described operations, all of the pairs of first valves, andall of the second valves, are simultaneously activated from stations 15and 16, respectively, as described above; thus, selective control ofcells into any individual microwell is controlled at the level of thewell-distribution network rather than by the valves controlling themovement of fluid within each passageway. This obviates the need forseparate control over the valves in each passageway. Although the abovecell-loading operations could be controlled by valving operations withineach passageway, it will be appreciated that the well-distributionnetwork offers a more efficient way of control fluid flow within eachpassageway. For example, in the present embodiment, controllingindividual first and second valves in all 16 passageways would require32 port stations rather than the 8+2 stations required with theconfiguration shown.

After loading each of the microwells with a selected number of cells,the device is switched to a cell-assay mode in which each of themicrowells in the device are exposed to selected cell-culture assayconditions. As one example, assume it is desired to assay the cells inthe two groups of eight wells with two different concentrations of thesame drug. Cell culture media containing each of the drug concentrationsare then placed in the reservoirs feeding port stations 2 and 3 in FIG.6A. By opening valves 106, 108 and closing vales 104, 105 in FIG. 6A,media from stations 2 and 3 are directed into the network sections 94,96, respectively. With the all of the network valves maintained in anopen condition, and with the first valves in the passageways maintainedin an open condition, and the second valves in closed condition, mediumfrom station 2 flows into each passageway, and around each microwell,exchanging medium components in each microwell across the porous barrierin each microwell. Over the course of the incubation period, the mediumin the microwells remains substantially equilibrated with that insidethe microwells, ensuring constant and uniform reaction conditions withinthe wells.

Also during the assay period, valves 114 and 116 are maintained in anopen conditions, allowing material being forced through the passagewaysto be collected at the reservoir services by port station 12.

At the end of the cell-assay periods, e.g., after a 1-2 day incubationperiod, material from the two sets of microwells may be collected intoeach of two collection reservoirs through stations 13 and 14. As seen inFIG. 6A, switching valves 114 and 116 to their closed condition willdivert media from the lower and upper sets of passageways, respectively,to stations 14 and 13, respectively, from which the material iscollected in corresponding reservoirs 13, 14 on plate 24. The collectedmaterial may then be assayed conventionally, for example, for drugmetabolic products or cell-secreted products.

Alternatively, where the cell assay involves inhibition or stimulationof cell growth, or uptake by the cells of a fluorescent material, thecells in the wells in each device may be inspected periodically, byremoving a selected plate from tower 28 in FIG. 1, placing the plate onstage 35 for microscopic viewing, and individually inspecting the cellswithin each microwell.

C. Valved Microfluidics Device and Microvalve

This section will describe the construction and operation of amicrovalve in accordance with one aspect of the invention, and a valvedmicrofluidics device employing one or more such microvalves. Themicrovalve is suitable for use in the array device already described,and the microfluidics device may contain one or more valved microwellpassageways of the type described above.

D. Microfabrication Methods

FIGS. 13-17 describe the various steps used to prepare multilayer,integrated devices useful for practicing the subject invention. Themicrofabrication techniques 25 used to prepare molds for the twoelastomer layers are described in FIGS. 13A-H and 14A-E. FIG. 15describes the semi-rigid plastic substrate used to support the upperlayer, FIG. 16 details the molding process and the elastomer monolithassembly. FIGS. 17A-D illustrates the holder, and the final assembly ofthe device. The particular methods described here are illustrative, andshould not be viewed as limiting the scope of the device or the processfor fabricating such a device. Numerous analogous materials, steps,dimensions and methods may be used, as is readily understood to thoseskilled in the art.

FIGS. 13A-13H illustrate the process for preparing a mold useful formaking a microfluidic device layer elastomer part. In FIG. 13A, a wafer,1300, is used as the base substrate for mold fabrication. A pattern ofthe microfluidic layer that will be made is shown in the inset, 1310.The layer pattern is repeated across the surface of the wafer asindicated by the repeated square regions on wafer 1300. The wafer usedis preferably silicon, and may be of any of the standard sizes used inmicrofabrication foundries. Note however that wafer-based fabrication isnot necessary, but is used for convenience. A 4″ wafer size allows asubstantial number of patterns to be repeated across the surface, and isa size that is commonly available at commercial facilities. Other wafermaterials may be used as desired, with the appropriate methods analogousto those described here employed as necessary. These fabrication stepsare well-developed in the art.

In order to more clearly focus on illustrating the fabrication process,the microfluidic pattern 1310 is a simplified pattern, yet one thatincludes enough features to provide for a valved microfluidic device ofthe type discussed above. As exemplified in the figure, an inlet area1312 and an outlet area 1314 are provided for access with the externalworld. Inlet channel segment 1316 leads from the inlet area 1312 to anintersection with a pair of first arm channels 1320 a and 1320 b, andsecond arm channel 1322. The pair of first arm channels 1320 a and 1320b later merge downstream and lead into outlet channel segment 1328,which ends at outlet area 1314. Second arm channel 1322 leads into well1324. The well 1324 communicates with the first arm channels via aseries of smaller passageways 1326 connecting the two features.

The pattern in the microfluidic layer also includes the recesses of thevalve regions in the first and second channel arms. The recesses 1330 aand 1330 b are defined on either side of each of the first arm channels1320 a and 1320 b, respectively. Likewise the recesses 1332 flank thesecond channel arm 1322. The recesses are voids into which the sidewalls of the channels (secondary membranes) may deflect when thepressure is changed at the primary membrane, and thereby operate as partof the valve. The recesses thus serve to define the valve region of thechannel, although to be functional the primary membrane needs to also beprovided at the same region. Note that the primary membrane is definedthrough the fabrication of the pneumatic control layer, described inFIG. 14.

First, the microfluidic passageways 1326 that connect the well 1324 withthe first arm channels 1320 a and 1320 b are defined on the substrateusing standard photolithography techniques. These passageways, alsoreferred to as perfusion channels, are of a smaller height, and thus aredefined first, in a step separate from the other fluidic channels andwells. Photoresist is spun onto the wafer 1300, softbaked, and the waferis irradiated through a reticle defined with the pattern as shown in theplan view of FIG. 13B. Typically a negative photoresist is used, suchthat the portion defining the channels is irradiated and the rest of thewafer is not: The photoresist is next developed to remove thenon-irradiated portion of the resist. The remaining resist may behard-baked. The resulting wafer and defined resist are depicted in thecross-sectional view of FIG. 13B. As mentioned above, standardphotolithographic techniques are employed, and suitable process details,well-known to those skilled in the art, are readily adapted from, forexample, S. O. Kasap, Principles of Electronic Materials and Devices, 2dEd., McGraw-Hili (2001). Note also that ‘maskless’ lithography may beused instead. The particular means of arriving at the fabricatedstructure is not critical.

Next, the wafer surface is etched away using a dry etching process toleave raised portions that define the perfusion channels 1326.Approximately 1 μm of the surface is etched away using a SF₆ plasma. Forexample, using a PlasmaTherm PK-12 RIE, at an RF power of 100 W and achamber pressure of 60 mTorr, the etch is performed for two minutes. Theresulting wafer is illustrated in the cross-sectional view of FIG. 13C.Thereafter the photoresist atop the raised silicon is removed by anoxygen plasma etch, as shown in FIG. 13D. Using the same model plasmaetcher, an oxygen plasma is generated at an RF power of 150 W, and achamber pressure of 100 mTorr, and the wafer is exposed for five minutesto remove the photoresist.

FIG. 13E shows the results of a second photolithography step. As before,a photoresist is spread across the wafer surface. Here, it is preferableto form the resist with a height greater than that of the raised silicondefining the passageways 1326. For example, if the passageways have aheight of 1 μm, then the photoresist should be spun on to give a heightof at least 1 μm. The photoresist is irradiated, typically through areticle, and developed to define the pattern of microfluidic channels,wells and recesses described above. The contours of the features areshown in the plan view of FIG. 13E as the dark portion. Thecross-sectional profile of FIG. 13E reveals the relative height of theresist and the previously defined passageways 1326.

With the photoresist in place, the remainder of the wafer surface isetched away (FIG. 13F). In this step of the process, a deeper etch isperformed in order to create a mold having a deeper channel and chamber.For example, a deep reactive ion etch of 50 μm is made in the waferusing Surface Technology Systems' Advanced Silicon Etching (ASE) System,alternating etching at an RF power of 12 W, SF₆ at 130 sccm andpassivating at an RF power of 0 W and C4F₈ at 85 sccm, for 20 minutes,according to the manufacturer's protocol. Again, the remainingphotoresist is removed using an oxygen plasma treatment, using the sameprocess as described for FIG. 13D. The resulting profile is revealed inthe cross-sectional view of FIG. 13G. The two different heights in thewafer surface correspond to the channel and well depth, and theperfusion channel depth.

Finally, the microfluidic layer mold is completed by coating theprocessed wafer with a fluorocarbon layer, as shown in FIG. 13H. Thewafer is, for example, exposed to a C₄F₈ plasma in the ASE System at 85sccm for ten minutes. The resulting fluorocarbon coating isapproximately 10 nm thick. The coating serves to prolong the life of themold by protecting the silicon features and also to improve liftoff ofthe molded elastomer from the mold due to it being a lower stictionsurface. The coating and improved liftoff is particularly useful for thesuccessful molding of elastomer parts that are thin, e.g. less than 100μm thick, or even about 70 μm thick. Note that where the channel depthhas been lithographically defined to be about 50 μm molding a 70 μmthick part means there is only a 20 μm portion adjacent to the channelor well.

FIGS. 14A-14E illustrate the process for preparing a mold useful formaking a device control layer elastomer part. In FIG. 14A, a wafer,1400, is used as the base substrate for mold fabrication. A pattern ofthe microfluidic layer that will be made is shown in the inset, 1410.The layer pattern is repeated across the surface of the wafer asindicated by the repeated square regions on wafer 1400. For conveniencethe same microfabrication methods and materials used for the first layerare also used here. The pattern is conveniently prepared at the samesize as that in the first microfluidic device layer in thisexemplification. Note that the first and second layers are to be alignedand bonded together in the final device. The channels and chambersdesigned in this second layer form the features that will operate withthe recesses and secondary membranes defined in the first layer to actas a pneumatically controlled valve. It is also contemplated that apneumatic-activated device control layer might only be needed to cover afraction of the microfluidic device layer, depending on the size,complexity and layout of the device channels. Accordingly, the wafersize and/or the pattern size may differ between the two layers.

As exemplified in FIG. 14A, the pattern 1410 contains three microvalves,with two being simultaneously active on one line. There is a first inletarea 1412, leading to paired first control channels 1414 a and 1414 b,and a second inlet area 1422 leading to a second control channel 1424.The inlet areas function to provide access with the external world, aswill be described later. The control channels 1414 a and 1414 b lead topneumatic chambers 1416 a and 1416 b, respectively. Likewise secondcontrol channel 1424 leads to pneumatic chamber 1426. The chambers aredefined to occupy a space directly above the valve region defined by therecesses in the fluidic layer mold. The channel and chamber systems inthis layer do not have an “exit”, but are closed end networks that willbe pressurized as needed to actuate the valve. Because the chamber 1416a and 1416 b are on the same line they will operate in concert, withboth being either open or closed.

The fabrication process follows that already described in conjunctionwith FIG. 13, except that there is only photolithography step for thecontrol layer mold. The photoresist is spin-coated, irradiated,typically through a reticle, and developed to define the pattern of thechannels and chambers described above. The contours of the features areshown in the plan view of FIG. 14B as the dark portion. Thecross-sectional profile of FIG. 14B reveals the distribution of thephotoresist.

The exposed wafer surface is etched away using a dry etching process toleave raised portions that define the control channels (1414 a, 1414 b,1424) and chambers (1416 a, 1416 b, 1426). Approximately 5 μm of thesurface is dry etched away using a SF₆ plasma, using the PlasmaThermPK-12 RIE, at an RF power of 100 Wand a chamber pressure of 60 mTorr,for ten minutes. The resulting wafer is illustrated in thecross-sectional view of FIG. 14C. Thereafter the photoresist atop theraised silicon is removed by an oxygen plasma etch, as shown in FIG.14D. Using the same process and equipment as described for FIGS. 13D and13G, an oxygen plasma is generated at an RF power of 150 W, and achamber pressure of 100 mTorr, and the wafer is exposed for five minutesto remove the photoresist. The resulting profile is revealed in thecross-sectional view of FIG. 14D.

Finally, the control layer mold is completed by coating the processedwafer with a fluorocarbon layer, as shown in FIG. 14E, using the sameC₄F₈ plasma etch described above, with the same resulting advantages.

FIG. 15 illustrates a semi-rigid sheet 1500 also used in the fabricationof a fluidic device. The sheet is, for example, an acrylic polymer, butalso may be chosen from any of polymethyl methacrylate (PMMA),polycarbonate, polystyrene, polynorbornene, polyethylene terephthalate,polyethylene, polypropylene and poly(4-methyl-1-pentene), or other likethermoplastic material, so long as there is an adhesion promoter bywhich the material can be bonded to the elastomer part. The sheet is tobe provided at the same size as the control layer part.

Preferably sheet 1500 is approximately 1.5 mm thick, such that itmaintains a two-dimension rigidity. The sheet is to perform (1) as abacking for the elastomer parts to (a) make handling easier and (b)prevent tearing, and (2) as a surface for bonding the elastomer deviceto a holder. Note that a thinner sheet may be used, but if the sheet nolonger maintains a two-dimensional rigidity the above purposes will notbe met. The sheet may be thicker, e.g. several mm's in thickness, ormore, though at greater thicknesses the extra material becomessuperfluous although not detrimental.

One surface of sheet 1500 is to be coated with an adhesion promoter1510. The promoter is to aid in the bonding of the sheet 1500 to themolded elastomer part, and is needed because of the dissimilarity of thematerials of the two parts. An exemplary adhesion promoter is the 1200Primer (Dow Corning, Midland, Mich.), which is typically used to promotethe adhesion of silicone materials to a variety of materials. Thispromoter is an appropriate choice given that the preferred elastomer isa silicone derivative, polydimethylsiloxane. Other promoters may beselected as appropriate for a given choice of materials for the sheetand the elastomer part, as are known to those skilled in the art ofadhesives and bonding of plastics and elastomers.

The adhesion promoter is to be applied to the surface of the sheet justprior to use. Manufacturer's recommendations should be followed as tothe timing of application prior to the bonding step for any promoterthat is selected.

FIGS. 16A-16K illustrate the molding process for the two layers, thebonding process putting the layers together, creation of the accessholes, and the bonding of the assembly to a bottom substrate.

First, as shown in FIG. 16A, the device layer elastomer part is preparedusing the mold 1600, as prepared by the steps of FIG. 13. The elastomerprecursor is spread over the mold using, for example, a spin-coatingtechnique. Other techniques for distributing the liquid over the moldsuch as dip coating, dispensing or roll coating may be used withoutlimitation. The elastomer precursor may be selected from the group ofmaterials such as precursors for polyisoprene, polybutadiene,polyisobutylene, polyurethane, silicone, and polysiloxane. Theprecursors may be liquid formulation of either monomers, oligomers orshort polymers that are capable of polymerization, furtherpolymerization or crosslinking and the like such that an elastomermonolith results. One preferable elastomer is a polydimethylsiloxane(generally referred to as PDMS). Numerous formulations for PDMSelastomers are commercially available, such as Sylgard 184 (Dow Corning,Midland, Mich.).

To prepare the device layer elastomer, for example, Sylgard 184 isdispensed over the wafer with mold, 1600, and by spin-coating, a layerof the PDMS precursor 1610 is formed with a height of 70 μm. The heightof elastomer ultimately obtained should be considered when determiningthe height of the precursor to be spread over the mold. For example, anyshrinkage or contraction that might occur when the precursor is curedshould be accounted for.

The height of the elastomer in relation to the height of the moldfeatures determines the thickness of the membrane between the channelfeature and the next layer of elastomer, the control layer, in the finalassembly. In locations where the channel portion is to be the valveregion, the membrane will be the primary membrane of the valve, and itsperformance and activation parameters will be determined by thethickness established in this step. A thickness of about 20 μm ispreferable, though the thickness may reasonably vary. As one skilled inthe art would know, a thinner membrane may not be as durable, but thevalve would be capable of activation at lower pressure. Conversely, athicker membrane would be more robust and durable, but would requiregreater operating pressures to be activated. Membranes thicker thanabout 100 μm generally require activation pressures that are high enoughto damage the material. Furthermore the thickness makes it difficult toactivate the valve into a fully closed position and thus is notpreferred where complete closure of the valve is necessary.

After spin-coating the PDMS precursor, the material is partially curedby treatment in a 60° C. convective oven for one hour. FIG. 16B showsthe mold 1600 with the partially cured elastomer part 1620 in place onthe mold.

In parallel, the control layer elastomer part is prepared with a similarmaterial. FIG. 16C illustrates the mold 1630, prepared as described inFIGS. 14A-E, and a fixed volume of the elastomer precursor dispensedover the mold 1640. The volume is determined by a calculation of thevolume of the part, given the mold topography and the desired thicknessof the part.

The semi-rigid sheet described in FIG. 15 is then placed over the top ofthe dispensed elastomer precursor with the side bearing the adhesionpromoter contacting the elastomer. For example, two methods forperforming this are illustrated in FIG. 16D. The sheet 1650 may besimply laid on top of the elastomer, whereby gravity and surface tensionwork to bring the sheet flat against the entire surface of elastomer1640. In another embodiment, the mold 1630 and elastomer 1640 may beplaced within a fixed height spacer fixture 1660, with the sheet 1650positioned over the edges of the spacer to ensure that the sheet liesparallel to the molded surface, and a molded part with a uniform, fixedheight consistent from part to part is obtained. For example, the mold1630 is placed within the fixed height spacer 1660. A fixed volumesufficient to form a 200 μm thick layer of Sylgard 184 is dispensed ontothe mold. Thereafter, a 1.5 mm thick acrylic sheet 1650, with anadhesion promoter coated on one side, is placed on top of the spacerfixture 1660 such that the promoter coating contacts and spreads theSylgard 184 out over the entire mold surface.

After preparing the assembly, the elastomer material is partially cured,again by treatment in a 60° C. convective oven for one hour. FIG. 16Eshows the mold 1630 with the partially cured elastomer part 1670 inplace on the mold, and the semi-rigid sheet 1650 affixed to the top ofthe elastomer part. When the assembly has cooled to room temperature,the now joined sheet and elastomer 1680 is peeled off of the mold, asshown in FIG. 16F. The flexible yet semi-rigid backing sheet permits theelastomer part to be readily peeled off the mold surface in an easymotion with little risk of tearing the control device layer elastomerpart.

The upper layer, the control device layer with the backing sheet 1650 isaligned over the device layer mold 1600 and molded part 1620 such thatthe operational features of the valves are properly positioned, and thencontacted together for bonding. This process may be performed with theaid of a standard mask aligner, such as a Quintel Q-4000 Series maskaligner. The assembly now comprises the lower mold 1600, the devicelayer molded part, the control layer molded part, and the semi-rigidsheet, as shown by the cross-sectional view of FIG. 16G. The elastomerparts 1682 of the assembly are then treated in a 60° C. convective ovenfor one hour to yield a cured and bonded part 1684, shown in FIG. 16H

When the assembly has cooled to room temperature, the now joined sheetand bonded, cured elastomer 1684 is peeled off of the mold, as shown inFIG. 16I. Again, the flexible yet semi-rigid backing sheet permits theelastomer part to be readily peeled off the mold surface in an easymotion with little risk of tearing the bonded elastomer parts. The mold1600 may be reused again, and the device assembly 1686 is taken forfurther processing.

Next, access holes are formed in the device 1686 by piercing, drilling,ablating, or laser cutting. The access holes are bored through theentire height of device 1686. The location of the access holescorrespond to the areas designated as the inlet areas or outlet areas ofeach of the two layers. Referring to the plan view drawing of FIG. 16J,there are four access holes formed in the device 1686, two that addressthe fluidic device layer (inlet, 1690; outlet 1692) and two that addressthe control layer inlets (1694 and 1696). The two access holes thataddress the control layer also appear in the cross-sectional view ofFIG. 16J.

The holes are preferably formed by laser cutting techniques. The lasermay be either a continuous wave (CW) mode or pulsed mode type. Althoughgreater care must be used when cutting with a CW laser because of thepossibility of overheating or charring the material, operation with CWlasers is possible so long as the device is no thicker than severalmillimeters. For example, a VersaLaser, from Universal Laser Systems(Scottsdale, Ariz.), operated at 25 W and controlled to move at 40% armspeed over steps of 1000 points per inch to cut the access holessuccessfully bores holes through devices comprising a 1.5 mm sheet and a˜300 μm elastomer monolith. The holes are cut by controlling the laserto follow a circular pattern around the center point of the holelocation via the software controls provided with the instrument. If athicker device undergoes charring during laser cutting with a CW laserthen operation with a pulsed mode laser is preferred.

The bottom surface of device 1686, bearing the molded imprint of thefluidic device layer, is next bonded to a rigid substrate to enclose thechannels and chambers of the fluidic layer, as well as to seal off thebottom of the access holes. The substrate preferably contains surfacehydroxyl groups to make the surface amenable to bonding with theelastomer material. For example, substrates such as glass, metal oxides,silicon with an oxide surface are suitable, providing both rigidity andthe necessary affinity for bonding to the elastomer. FIG. 16K shows aglass substrate 1696 bonded to the device 1686 to form the functionaldevice 1698. The glass substrate 1696 is precut to the size of thedevice. Then, both the glass surface and the PDMS surface of device 1686are each treated with an oxygen plasma using the PlasmaTherm PK-12 RIE,at an RF power of 40 W and a chamber pressure of 2 Torr for 40 seconds.After oxygen plasma treatment the two pieces are aligned and contacted.As a result of the treatment the two surfaces will bond together to makea leak-proof seal.

The device assembly 1698 is fully capable of use as a microfluidicdevice with functioning pneumatically controllable valves. For greaterease of use and to enable interfacing the device with automated roboticsystems, the device 1698 may be integrated with a housing or a holderthat facilitates putting reservoirs of fluids in communication with thedevice, and connecting fluid lines and pneumatic controls to the device.When low pneumatic pressure is required, gravity-driven flow can be usedby tilting the device 1698.

FIGS. 17A-17D illustrates the design and fabrication of a holder thatintegrates with the microfluidic device and serves to facilitate accessfrom the external world to the fluidic and control layers inside thedevice. The holder is assembled from two separate parts. The upper part1700 is illustrated in FIG. 17A. This upper part comprises a body with aplurality of holes opened through from the top surface to the bottomsurface. The side wall of each hole will form the side of a reservoir orconnector port in the completed holder. The part also comprises aplurality of shallow cavities at the bottom surface of the part. Thecavity openings may be circular or rectilinear or a combination thereofwith respect to the bottom surface. Generally, the depth of thesecavities is uniform, though uniformity is not required. One cavity isprovided adjacent to each hole, positioned to intersect with a side wallof the adjacent hole. Thus, the top surface has a roughly circularopening, and the bottom surface has an eccentric opening, defined by thecombined area of the hole and the cavity.

The holes may be shaped for a variety of special purposes. For example,where the hole is to be used as a liquid reservoir, the hole may bedimensioned to be that of a standard microwell plate, e.g. a 96-wellplate or a 384-well plate or other sizes. The standard sizes are setforth in the SBS standards and can be found onwww.sbsonline.org/msdc/pdf/text1999-04.pdf. Also, the holes themselvesmay be positioned relative to one another at standard distances thatconform to SBS standards. The purpose of using standardized well shapesand distances is to facilitate interfacing the part with automateddispensing and handling systems. The hole may also function as aconnection port, e.g. for receiving the tips, couplers or connectors offluid (liquid or gas) lines. There are numerous standard interfaces forsuch connections, and any of these may be employed in the design of thepart. One common example of a connection is a Luer connector, thoughmany others are possible. The hole, shaped as a Luer receiver, wouldpermit the rapid insertion of a Luer-tipped syringe body. Note also thatthe various holes may be prepared with differently shaped or sizedholes, depending on the purpose of the hole and the underlying accesshole in the fluidic device with which it communicates.

The part itself may be made of any suitable durable material, with metalor plastic being preferred. The part may be machined, cast or molded,according the material type chosen. Molded plastic parts are preferredfor their low cost. Standard injection molded methods are suitable formaking the part, being able to provide the necessary precision anddetail of shape desired.

FIG. 17B shows the lower part 1710 of the holder body. As is apparent inthe plan view, the upper surface comprises a plurality of holes. Theseholes are positioned to align with the cavities formed in the lowersurface of upper part 1700, with little or no overlap with the openingof the holes in the upper part. The lower surface has a cavity sized inall dimensions to house the microfluidic device 1698. The holes in theupper surface lead into the cavity opened in the lower surface, asillustrated in the cross-sectional view of FIG. 17B. Also, the uppersurface of the lower part largely covers the hole opening in the bottomof the upper part, thus forming in the completed part the bottom of areservoir defined by the hole in the upper part.

As shown in FIG. 17C, the two parts are mated, with the upper part'scavities and the lower parts' holes aligned, and joined by, for example,thermal diffusion bonding, adhesive bonding using a pressure sensitiveadhesive, solvent welding, or other technique for joining two plasticsas is known in the art, to give the completed holder 1720. Thus, upperpart 1700 and lower part 1710 were formed of acrylic by injectionmolding. The parts were then aligned and bonded using a Carver Lab Pressto apply 0.5 tons of force, while heating the surface to 106° C. andholding for 10 minutes.

Note that the number of holes in the upper part generally correspond tothe number of access ports formed in the fluidic device (e.g. 1698). Theholes in the upper part 1700 communicate with the access holes, but aregenerally not positioned directly vertically above the access holes,although if the space permits they may be so positioned. Often, due tothe density of access holes in the design, the larger holes used in theholder 1730 need to be offset from a position vertically above theaccess holes. The two-part holder design shown in FIG. 17 isillustrative of one means to achieving such a design for larger externalports, reservoirs and connectors to communicate with the smaller accessports to the internal network of channels and chambers in the fluidicdevice.

The last step in assembling the integrated microfluidic device is shownin FIG. 17D. The microfluidic device 1698, is positioned within thecavity of the lower part of holder 1720, with the semi-rigid sheet 1650contacting the holder. An adhesive is conveniently applied to join thetwo parts. For example, where the holder and the sheet are both acrylic,an acrylic cement, IPS-3, from TAP Plastics (Stockton, Calif.), is usedto join the parts to give integrated device 1730.

The design of the microfluidic device used to illustrate the fabricationprocess was simplified for the ease of presentation and explanation. Thesteps used in the process are generally applicable to a wide variety ofdevice configurations, including devices of much greater size,complexity and density, as would be appreciated by those skilled in theart of fluidic device design and fabrication. For example, the devicesdescribed throughout this disclosure are all capable of fabrication bythese methods.

What is claimed:
 1. A valved microfluidics device, comprising: asubstrate, a microchannel through which liquid can be moved from onestation to another within the device, and a pneumatic microvalve adaptedto be switched between open and closed states to control the flow offluid through a microchannel, said microvalve comprising: two or moreflexible membranes forming a valved region of the microchannel, the twoor more flexible membranes comprising a primary membrane forming a firstwall portion of said valved region and one or more secondary membranesforming one or more second wall portions of said valved region, said oneor more second membranes each joined to said primary membrane at acommon edge, and a chamber formed in said substrate, separated from saidmicrochannel by said primary membrane and adapted to receive a positiveor negative fluid pressure, thus to deform said primary membrane,wherein deformation of said primary membrane causes said one or moresecondary membranes to deform along said common edge and the combineddeformation of the primary and secondary membranes is effective toswitch the condition of the valve between its open and closed states. 2.The valved device of claim 1, which further includes, for each secondarymembrane, a recess formed in the substrate, into which the secondarymembrane is deflected when deformed.
 3. The valved device of claim 1,wherein the primary membrane comprises a top-wall portion of said valvedregion and the one or more secondary membranes comprise a pair ofopposite side-wall secondary membranes, wherein the valved region ofsaid microchannel, with the valve in its open state, is substantiallyrectangular.
 4. The valved device of claim 2, wherein application ofpositive pressure to said chamber, to place the valve in its closedstate, causes the primary membrane to bow outwardly into said channel,and the secondary membranes to bend outwardly at their common edges withthe primary membrane, into the associated recesses, thus to enhance theextent of sealing between the primary membrane and the two secondarymembranes as the primary membrane is deformed.
 5. A microfluidic devicecomprising: an elastomeric monolith having a first and a second surface,microfluidic channels disposed in the first surface, and pneumaticcontrol chambers disposed within the body of the monolith, themicrofluidic channels further comprising at least one valved region, theat last one valved region comprising a primary membrane and one or moresecondary membranes, the one or more secondary membranes joined to theprimary membrane at a common edge; a rigid substrate bonded to the firstsurface and enclosing the microfluidic channels disposed therein; and asemi-rigid substrate bonded to the second surface of the elastomericmonolith; wherein the pneumatic control chambers are configured toreceive a positive or negative fluid pressure to deform said primarymembrane, wherein deformation of said primary membrane causes said oneor more secondary membranes to deform along said common edge and thecombined deformation of the primary and secondary membranes switches thecondition of the valved region between opened and closed states.
 6. Thedevice of claim 5, wherein said elastomeric monolith is assembled from afirst elastomeric layer having microfluidic channels and a secondelastomeric layer having pneumatic control chambers.
 7. The device ofclaim 5, wherein said semi-rigid substrate is a thermoplastic polymersheet comprising a material selected from the group consisting ofacrylic, polymethyl methacrylate, polycarbonate, polystyrene,polynorbornene, polyethylene terephthalate, polyethylene, polypropyleneand poly(4-methyl-1-pentene).
 8. The device of claim 5, wherein saidelastomeric monolith comprises a material selected from the groupconsisting of polyisoprene, polybutadiene, polyisobutylene,polyurethane, silicone, polysiloxane, and polydimethylsilozane.
 9. Thevalved device of claim 1, wherein said one or more secondary membranesare positioned perpendicularly to said primary membrane.
 10. The valveddevice of claim 2, wherein each recess formed in the substrate isadjacent to its secondary membrane and spaced opposite from themicrochannel.